Blood group serology requires the determination of blood cell compatibility between donor and patient before a transfusion or organ transplant. Blood cell compatibility is determined by the non-occurrence of an immunological reaction between antibodies contained in the blood serum of a patient and antigens present on blood cells from a donor. A patient whose red blood cells are group A, i.e. having "A" antigens on the red cells, will have Anti-B antibodies in his or her serum. Thus, if such a person is given type B blood, an immunological reaction will occur with possible serious clinical consequences.
Tests for blood cell typing and compatibility are generally of two types: (1) agglutination tests which determine whether a specific antibody added to the cells will cause their agglutination, and (2) cell lysis tests which determine whether a specific antibody added to the tested cells together with serum complement results in hemolysis.
In blood cell typing and compatibility test procedures commonly used, both agglutination and cell lysis tests are carried out either manually by a trained technician or using automated devices. However, these tests are occassionally not sensitive enough to detect some weakly reacting antibodies or to differentiate complex mixtures of antibodies.
Enzymes are catalytic proteins which accelerate certain biological reactions. In immunohematology, proteolytic enzymes such as ficin, bromelin, papain, and trypsin remove proteins from the red cell surface. Red cell agglutination may be observed after enzyme treatment due to the greater exposure of antigen sites and/or the reduced surface charge of the red cells. This may result in a decreased cell-cell repulsion, or, enhanced antibody uptake. Certain other antigen-antibody reactions may be depressed or eliminated, due to the removal of some antigen sites after this enzyme treatment.
Enzymes can be used in two different ways in blood group serology. An enzyme solution can be added directly to a serum-cell mixture (one stage method), or the red cells can be pretreated with enzyme before serum is added (two stage method). The one stage method is faster and more convenient, but is not as sensitive as the two stage method. An advantage of the latter is better modification of the red cell membrane due to the absence of serum proteins.
When an unexpected blood group antibody is detected in a patient or donor serum, it is important to determine its specificity. The use of enzyme treated cells can be of help in determining the identity and clinical significance of the antibody. This is especially true when there are multiple specificities or the antibody has weak reactivity. Enzyme techniques strengthen the reactions seen with many blood group antibodies, especially those in the Rh and Kidd systems. Antigens in the MNS and Duffy systems are usually destroyed, so reactivity of antibodies directed against these antigens will be depressed. Standard enzyme modification techniques are described in the American Association of Blood Banks 1985, Technical Manual, 9th Ed., Arlington, VA, pp. 560-587.
One example of a diagnostic application of enzyme-treated cells is set forth in U.S. Pat. No. 4,275,053 (Rosenfield et al.). This patent discloses a solid-phase blood typing procedure based upon either agglutination or immune lysis. In this invention, a monolayer of cells is irreversibly bound to a solid matrix. A serum containing antibodies is brought into contact with the bound cell layer. Immuno-adsorption of antibodies by the bound cells occurs where the antigens of the cell membranes and the antibodies in the serum are complementary to each other. The antibody-sensitized monolayer of blood cells can either bind a second layer of blood cells carrying complementary antigen (solid-phase agglutination) or undergo lysis in the presence of serum lytic complement (solid-phase immune lysis). The results are evaluated quantitatively by such standard procedures as densitometric scanning or radioisotopic counting. It is taught at col. 12, lines 9-27 that the cells used in the Rosenfield et al. invention may be treated. For example, erythrocytes (red blood cells) may be treated with agents which potentiate agglutination such as colloids, proteases, polyelectrolytes, and buffer systems. The use of bromelin, a protease is disclosed. It is noted at col. 12, lines 35-41, that protease treatment may destroy the antigen to be assayed and "a very light treatment" is therefore taught.
Another example may be found in U.S. Pat. No. 4,608,246 (Bayer et al.). This patent discloses a method of testing the ABO forward blood grouping wherein a known antibody is attached directly to a solid surface and an unknown blood sample is activated by treating the sample with an effective quantity of a proteolytic enzyme. The activated sample is then contacted to the solid surface and any antigens specific for the known antibody will undergo immune reaction with the resulting adhered red color on the surface providing an indication of the presence of specific antigens. The invention also contemplates a method of testing applicable to reverse or serum ABO blood grouping. A solid surface capable of supporting an immunological reaction is provided and known antigens are adsorbed on the surface. The surface is then contacted with an unknown blood component which may contain unknown antibodies specific for the known antigen previously attached, in which case an immune reaction will occur. A solution of red blood cells containing the known antigen first attached to the solid surface is then activated by treating it with an effective quantity of a proteolytic enzyme. This known, activated solution is then brought into contact with the solid surface and the known antigens will undergo an immune reaction to the extent antibodies specific thereto were present in the blood component. Any resulting immunologically adhered red color on the surface will indicate the presence of these specific antibodies in the blood component. In carrying out the method of the Bayer et al. invention, it was found that considerable advantages resulted when the blood component is activated with a proteolytic enzyme which is characterized by the ability to modify red blood cells to enhance their serological activity. Examples of suitable proteolytic enzymes for this purpose include bromelin, papain, trypsin, ficin, proteinase K and pronase. A one step bromelin method is preferred.